Optional Protocol for Running QC Bulk Species-Mixing RNA-Seq using ChronoSeq or Dropseq beads¶

Solutions and Buffers to Make¶

  • 1M DTT:
    • Dissolve 1.55g solid DTT in 10ml of Deionized Water. Hazardous Chemical Read SDS. Work in Fume Hood

More economical to make yourself from Solid DTT. Make 50ml or more at a time and store in 10ml Aliquots for upto an year. DTT is not stable at room temperature. Make this solution quickly and store at -20°C, unless you plan to use it immediately to make another Buffer.

  • 5X RT Buffer:
    • 250mM Tris-HCl (pH 8.3 at 25°C)
    • 375mM KCl
    • 15mM MgCl2
    • 50mM DTT

More economical to make yourself for large volumes. Make 100ml of more at a time and store in 11ml Aliquots at -20°C for upto an year. DTT is not stable at room temperature. Make this solution quickly and freeze.

Preparation a Day Before¶

  • Turn on the UV for the Cell culture hood and wait 15 minutes.
  • Aseptically prepare 4 eppendorf tubes with 10μl for each bead type, condition or timepoint the day before. Bead concentration should be 450beads/μl:
    • Spin down the beads in the Tube in which your beads are stored.
    • Vortex this Tube at Maximum speed for 3 seconds to make sure the beads are evenly suspended before transfering them.
    • There should be 4 eppies for each type of bead/timepoint/condition.
    • Two eppies should be labeled with H(Human) and Name of Condition/Bead type/Timepoint.
    • The remaining two should be labeled with M(Mouse) and Name of Condition/Bead type/Timepoint.
    • For example if there are two conditions there should be 8 total eppies each with 10μl of beads.

Mix Beads with Cell Lines¶

  • Prepare enough 40μm filtered 6X SSC Buffer for the experiment. You will need 1ml of 6X SSC buffer per eppie. Keep this buffer at room temperature.
  • Prepare enough 2X RT buffer from 5X Stock for the experiment. You will need 2ml of 2X RT buffer per eppie. Put this buffer on Ice.
  • Using the Protocol for Preparing Cells make Cell Suspensions for 3T3 and HEK293 at 600 cells/μl for both. Do not mix the cell lines, prepare a separate tube with 1ml each.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Add the Cell suspension to the beads and mix well:
    • There should be four eppies for each condition. Two for Human Cells and Two for Mouse Cells.
    • Mix 8μl of Mouse Cells(3T3) for the M(Mouse) eppies. Pipette up and down several times to mix well.
    • Then quickly put these eppies on ice.
    • Mix 8μl of Human Cells(HEK293) for the H(Human) eppies. Pipette up and down several times to mix well.
    • Then quickly put these eppies on ice.

Wash Beads with 6X SSC and 2X RT Buffer¶

  • Wash each eppie once with 1ml of 6X SSC and twice with 1ml of 2X RT buffer:
    • Add 1ml of room temperature 6X SSC buffer. Vortex the beads as full speed for 2 seconds.
    • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
    • Carefully remove 900μl of Liquid without disturbing the beads.
    • Add 1ml of 2X RT buffer. Vortex the beads at full speed for 2 seconds.
    • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
    • Carefully remove 1ml of Liquid without disturbing the beads.
    • Add 1ml of 2X RT buffer. Vortex the beads at full speed for 2 seconds.
    • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Label a new eppies with the Condition/Bead-Type/Timepoint and date of the experiment. For example, if there are two Conditions/Bead-Types/Timepoints then you will need two new eppies.
  • Carefully remove 900μl of 2X RT buffer from each eppie without disturbing the beads.
  • Now combine all 4 Eppies(Human+Mouse) for a single Condition/Bead Type/Timepoint into the new Eppies.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Follow the Library Preparation Protocol from this point onwards.

Protocol for Running Bulk Time-Series RNA-Seq using ChronoSeq beads¶

Solutions and Buffers to Make¶

  • 1M DTT:
    • Dissolve 1.55g solid DTT in 10ml of Deionized Water. Hazardous Chemical Read SDS. Work in Fume Hood

More economical to make yourself from Solid DTT. Make 50ml or more at a time and store in 10ml Aliquots for upto an year. DTT is not stable at room temperature. Make this solution quickly and store at -20°C, unless you plan to use it immediately to make another Buffer.

  • 5X RT Buffer:
    • 250mM Tris-HCl (pH 8.3 at 25°C)
    • 375mM KCl
    • 15mM MgCl2
    • 50mM DTT

More economical to make yourself for large volumes. Make 100ml of more at a time and store in 11ml Aliquots at -20°C for upto an year. DTT is not stable at room temperature. Make this solution quickly and freeze.

Aliquot small samples of beads into 12 eppies a day before¶

  • Prepare 12 eppendorf tubes with 10μl for each bead timepoint/Seq Number the day before. Bead concentration should be 450beads/μl:
    • Spin down the beads in the Tube in which your beads are stored.
    • Vortex this Tube at Maximum speed for 3 seconds to make sure the beads are evenly suspended before transfering them.
    • There should be 12 eppies for each timepoint/Seq Number (SEQ1-SEQ12).

Mix Beads with Cell Suspension Sample¶

  • Prepare enough 40μm filtered 6X SSC Buffer for the experiment. You will need 1ml of 6X SSC buffer per eppie. Keep this buffer at room temperature.
  • Prepare enough 2X RT buffer from 5X Stock for the experiment. You will need 2ml of 2X RT buffer per eppie. Thaw and make this buffer from stock right before the washing steps below.
  • Spin down the eppies with the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Add 0.25μl RNAase Inhibitor and 0.5μl 200mM DTT to each eppie and keep on ice.
  • At the same time prepare a cell suspension by choosing the best protocol appropriate for your cell type from here.
  • Keep the cells suspendend at 37°C with magnetic stirring inside a 50ml Falcon Tube with 5%CO2 either using the ChronoSeq device or inside a lab incubator. If using an incubator make sure the tube is vented.
  • Perturb your cells. However, always take one sample before perturbing your cell suspension with SEQ1 as a baseline.
    • Add the 8μl of cell suspension @600cells/μl at the desired time interval to the corresponsing Seq Number for ChronoSeq beads and mix well.
    • For example for Cell Sample number 1, mix with Seq 1. For Cell Sample number 5 mix with Seq 5 etc..
    • Pipette up and down several times to mix well.
  • Immediately after mixing put the beads on Ice. Keep the beads on Ice till all the Cell Samples have been collected.
    • Put the Tubes down deep into the ice.
    • Compress the Ice to make sure the bottom of the tubes remain in constant contact with the ice.
  • Thaw reagents needed for the RT reaction/library prep and washing steps below.

Wash ChronoSeq Beads with 6X SSC and 2X RT Buffer¶

  • Wash each eppie once with 1ml of 6X SSC and twice with 1ml of 2X RT buffer:
    • Add 1ml of room temperature 6X SSC buffer. Vortex the beads as full speed for 2 seconds.
    • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
    • Carefully remove 900μl of Liquid without disturbing the beads.
    • Add 1ml of 2X RT buffer. Vortex the beads at full speed for 2 seconds.
    • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
    • Carefully remove 1ml of Liquid without disturbing the beads.
    • Add 1ml of 2X RT buffer. Vortex the beads at full speed for 2 seconds.
    • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Label a new eppie with a Brief Description and date of the Experiment.
  • Carefully Remove 1000μl of 2X RT buffer from each eppie without disturbing the beads.
  • Now combine all timepoints/seq numbers into the new Eppie using a P200.
  • Spin down the beads using a tabletop centrifuge @ 2500xg for 1 minute.
  • Follow the Library Preparation Protocol from this point onwards.